The advantage of pyrazinamide(PZA) therapy in rendering tissues of tuberculosis infected mouse to be "non-culturable" lead to its designation as "sterilizing" agent which was translated clinically to lasting cure for patients at the shortest course of therapy. However, PZA activity in vitro is relatively poor and non-bactericidal. The explanation for this apparent paradox should be sought in enhancement of its mechanism of killing in-vitro upon in vivo conditions (such as activated macrophages). Since PZA is mainly effective when given in the early phase of tuberculosis therapy and all culture tests methods for PZA susceptibility advocate and rely on the usage of replicating bacilli, PZA like other drugs should be studied with replicating bacilli.

PZA activity against mycobacteria depends on conversion to pyrazinoic acid (PA) by pyrazinamidase (PZAase). M. tuberculosis is susceptible to PZA only at acidified medium. The unique activity of PZA against M. tuberculosis amongst mycobacteria is due to an inefficient energy dependent efflux. The three factors conversion to PA (indispensible) acidic pH and deficient efflux combine, and result in "huge" intracellular accumulation of PA, which did not affect the intracellular pH.

PZA analogs were synthesized; their activity does not rely on PZAase conversion to PA. Many of the different PZA analogs have much higher potency against M. tuberculosis and also an expanded spectrum against other species of mycobacteria. PZA and its analogs likely share the same cellular site between mycobacteria species and PZA derivatives as such interaction is highly specific and essential cellular functions are not redundant.

The requirement of acidic pH for PZA activity can be markedly diminished by increased PZAse activity (converting PZA resistant M. smegmatis to PZA susceptible), by inhibition of efflux such as by reserpine and the analogs 5 chloro PZA and n-propyl PA, are active in neutral pH. The analog 5-Cl-PA is a stronger acid yet a weaker antimycobacterial agent than PA. Thus, acidity of the medium, merely a condition for PA accumulation, cannot have a role for the killing effect of PZA and intracellular acidification by PA is unlikely either. The dependence of PZA activity upon PA accumulation suggests that it affects an intracellular function.

PA and PZA analogs at equivalent dose (folds of the minimal inhibitory concentration, MIC) have similar antimycobacterial effect or killing curve. The antimycobacterial effect of PA and PZA analogs in replicating bacilli correlates with inhibition of fatty acid biosynthesis mediated by the multi-domain ,multifunctional enzyme fatty acid synthase I ( FAS I). These compounds inhibit FAS I in vitro (albeit at high concentration for PA and PZA), and were found to interact with FAS I enzyme in saturated transfer (STD)NMR studies.

June 1, 2011Day 2Session 1What Mice Tell Us About the Essentiality of PZAEric Nuermberger

In our experimental system, the addition of pyrazinamide, nicotinamide, pyrazinoic acid, nicotinic acid or isoniazid to uninfected J774 cells did not induce a proinflammatory cytokine response, and the addition of these compounds to M. tuberculosis-infected J774 cells did not alter cytokine secretion levels or patterns.

In the same experimental system, very few gene expression changes were observed with the addition of the above-stated drugs to either uninfected or infected J774 cells.

Administration of PZA 25 days by oral gavage was associated with a proinflammatory cytokine response in the lungs of both uninfected and M. tuberculosis-infected C3HeB/FeJ mice.

Think outside the box: PZA may have activity against other microorganisms.

Leishmaniasis is a chronic, emerging parasitic disease caused by the protozoan parasite Leishmania. Clinical signs range from mild cutaneous lesions to systemic disease leading to death.

PZA has activity against Leishmania in vivo in both models of cutaneous and visceral disease.

Some PZA analogs (i.e. 5-chloro and 5-fluoro) had greater activity than the parental compound 5.The target for PZA in Leishmania is unknown, but our two main hypotheses are that the drug inhibits the activity of either microsomal elongases or sirtuins in this parasite.

What do we know about the intracellular environment experienced by M. tuberculosis?David Russell

PZA as a structural analog of nicotinamide could interfere with NAD+ salvage pathway. which uses nicotinamide to synthesize NAD+.

PZA could enter the NAD+ salvage pathway and either form a PZA-analog of a NAD+ metabolite and inhibit NAD+ biosynthesis or form a PZA-analog of NAD+ that could inhibit enzymatic reactions using NAD+ as cofactor.

Since PZA has been shown to have an effect on NAD+ concentration in eukaryotes, these hypotheses deserve to be studied.

Session 3In search of parsimony: An alternative hypothesis for the mode of action of pyrazinamideAnthony Baughn

Like rifampin, pyrazinamide is a sterilizing drug that accelerates the killing of "persisters" and is responsible with rifampin for the shortening of the TB treatment duration from 18 months to 6 months.

Pyrazinamide is often considered bactericidal against actively replicating tubercle bacilli.

In fact, we demonstrated that pyrazinamide has clear-cut activity against nonreplicating bacilli but no activity at all against actively replicating bacilli in the nude mouse model.

These findings support the hypothesis that accumulation of toxic products of pyrazinamide metabolism within nonreplicating bacilli is the mechanism by which pyrazinamide kills tubercle bacilli.

These findings raise the important question of why pyrazinamide is used during the initial 2-month phase of TB treatment and not during the continuation phase.

Efficacy of PZA administered as a single drug in various TB mouse infection models (GKO, Balb/c and Kramnik)Anne Lenaerts

PZA is a frontline drug that has unique sterilizing activity involved in shortening treatment of TB and MDR-TB, as it kills persister TB bacilli that are not killed by other TB drugs and is essential or irreplaceable in any new drug combination for treatment of TB.

PZA is a paradoxical TB drug that has no activity against TB bacilli in vitro at neutral pH when bacteria are growing but is only active at acid pH. The activity of PZA is enhanced under stress conditions such as starvation, hypoxia, energy inhibitors, etc, and is completely opposite to common antibiotics.

We identified a new target of PZA RpsA that binds to POA where overexpression of RpsA conferred 5-fold PZA resistance in MTB and a low level PZA-resistant M. tuberculosis DHM444 without pncA mutations carried an alanine deletion due to 3-bp (ΔGCC)missing in the C-terminus of RpsA.

The active form of PZA, pyrazinoic acid (POA), at therapeutically relevant concentrations (50-100 ug/ml), inhibited the trans-translation function of RpsA mediated through inhibition of tmRNA binding to RpsA.

Trans-translation is to rescue stalled ribosome facilitate the removal of deleterious partially translated proteins by adding a tmRNA encoded peptide tag recognized by proteases for degradation during stress conditions for cell survival. Trans-translation is dispensable during normal growth but becomes critical under stress conditions when bacteria stop growing, and POA inhibition of trans-translation is likely to affect the survival of non-growing persisters. Our study validates the trans-translation which is present in all bacteria but absent in eukaryotes as a critical target for developing persister drugs not only for TB but perhaps for other persistent bacterial pathogens.

Pyrazinamide (PZA) has potent sterilizing activity and is a highly important drug in current anti-tuberculosis (TB) combination therapy. Unfortunately, while PZA resistant TB has been increasing worldwide, rapid and reliable diagnostic tools for the detection of PZA-resistant TB are still unavailable. This presents a major barrier for treatment, especially for multi-drug resistant (MDR) and extensively drug resistant (XDR) disease. PZA is the least understood anti-TB drug due to its complex mechanisms of action and obstacles in establishing animal models for PZA testing.

Purpose

To review current PZA use and historical data as well as the resistance patterns and clinical correlations in different regions; to address and identify the most promising research approaches for developing accurate and feasible PZA susceptibility testing. Mechanisms of action, how to determine optimal use in new combinations for drug-susceptible and -resistant TB, novel treatment strategies, and approaches to improve activity and overcome toxicity and resistance will also be discussed.

To bring awareness to the scientific community regarding these research areas and to help identify the best approaches, strategies, and models for future advances in PZA research.

To discuss future directions on PZA research and establish joint efforts and partnerships between government, industry, academia, and non-profit organizations.

Who should participate in the workshop

The majority of the invitees will be basic and clinical researchers from the US and the countries that have a high TB burden. Ideally, this meeting will also include scientists from related areas of research, e.g., geneticists, microbiologists, and immunologists who have not previously conducted PZA research or any TB research.

Networking and discussions from the meeting are expected to result in knowledge exchange and foster further collaborations and joint research projects to advance the field.

A meeting report summarizing the state of the art in research on PZA and recommendations for future research will be composed. This report will be submitted for scientific publication to increase awareness in the scientific community and promote cutting-edge research.

The research priorities identified from this meeting may serve as a source for the development of a a) Program Announcement or another type of NIH FOA to support needed research and b) refined CDC and FDA research agenda.

Number of Participants: 70 to 100 people will be invited to participate in this meeting.